Research Fractionation of Natural Organic Matter by Size Exclusion Chromatography-Properties and Stability of Fractions MARGIT B. MU ¨ LLER, DANIEL SCHMITT, AND FRITZ H. FRIMMEL* Department of Water Chemistry, University of Karlsruhe, Engler-Bunte-Ring 1, D-76131 Karlsruhe, Germany
An aquatic natural organic matter sample was concentrated by membrane filtration and fractionated using a size exclusion chromatography (SEC) system with a Superdex 75 column. Stability over time was investigated by reinjection of selected fractions. The reinjection yielded single peaks of a more or less Gaussian shape. From the peak shape of the individual fractions it was concluded that nonSEC mechanisms play an important role for the elution behavior. No significant change in the UV chromatograms could be detected over 5 weeks, suggesting a high stability of the fractions and only little or no alterations of the molecular weight distribution during that time period. The fractions were reexamined using a liquid chromatography system with online dissolved organic carbon detection (LCDOC system) with a TSK HW-50 (S) column. The average total recoveries of all fractions were 90 ( 4% for the Superdex system (calculated from UV-peak areas), and 102% and 105% for the spectral UV-absorbances (UVA) and dissolved organic carbon (DOC) contents measured with the LCDOC system. Peak molecular weights (Mp) were determined using polystyrene sulfonate (PSS) standards. The Mp ranged from Mp ) 17870 ( 113 Da to Mp ) 799 ( 28 Da for the original sample and from Mp ) 17998 ( 306 Da to Mp ) 737 ( 30 Da for the selected fractions.
Introduction The term natural organic matter (NOM) is generally used for a whole group of water soluble compounds of a rather complex nature and a broad range of chemical and physical properties. To gain more information on the NOM composition, various fractionation methods have been used which are either based on chemical and physical properties (variation of pH value, salting-out effect), on charge characteristics (electrophoresis, ion-exchange), on the adsorption behavior (XAD8-procedure), or on the molecular size (sizeexclusion chromatography, ultrafiltration, flow field-flow fractionation) of the solutes (1). While size-exclusion chromatography (SEC) is a standard technique for the purification, or fractionation, of protein and peptide samples, it has not been used to the same extent for preparative NOM fractionation. This might in part be due to the fact that the sample composition in the latter case * Corresponding author phone: +49 721 608 2580; fax: +49 721 699154; e-mail:
[email protected]. 10.1021/es000076v CCC: $19.00 Published on Web 10/21/2000
2000 American Chemical Society
is not exactly known and that it is impossible to select an individual, well-defined substance which is to be isolated. In contrast, size exclusion chromatography has been used extensively to study the molecular size distribution of NOM samples. The interpretation of the results, however, is limited by the fact that it is rather difficult to obtain a separation based solely on molecular size. Many authors, among them Gelotte (2), Lindqvist (3), Swift and Posner (4), Aho and Lehto (5), Fuchs (6), and Specht (7) have reported nonspecific, i.e., ionic and adsorptive interactions between the gel and NOM solutes. Those limitations must be kept in mind when interpreting SEC results. Another problem is the lack of suitable standards for the molecular weight (MW) determination of NOM, that is, standards which have the same structure and properties as NOM components. Polystyrene sulfonates (PSS) are thought to be suitable standards for the determination of the MW of NOM samples due to the assumed structural similarities. However, they will only provide approximate values, a fact which has been pointed out by authors such as Perminova et al. (8), Peuravuori and Pihlaja (9), and Pelekani et al. (10). In our study the determination of molecular sizes was of minor interest, though. The main purposes of this work were (1) to prepare fractions of a natural organic matter sample, (2) to determine recoveries for the fractionation procedure, (3) to investigate the stability of the fractions over time, (4) to reexamine the fractions using an independent analytical SEC system, and (5) to compare the results obtained with the two systems.
Materials and Methods Sampling and Sample Preparation. An aquatic natural organic matter sample was taken from the Hohlohsee, a bog lake in Southern Germany, and filtered through a 0.45 µm cellulose nitrate membrane. The sample was concentrated by ultrafiltration using a polyethersulfone membrane (UFPES 4 H, Hoechst, Germany) with a nominal MW cutoff of 4000 Da. Details on the sampling and ultrafiltration procedures are given by Frimmel and Abbt-Braun (11). The concentrate, HO12K, represents 65% of the original organic carbon content. Before fractionation, HO12K was diluted with MilliQ-water (Amicon/Millipore, USA) with a dissolved organic carbon (DOC) content of β(DOC) ) 0.2 ( 0.02 mg/L to a β(DOC) ) 180 ( 7 mg/L (LCDOC bypass measurement, number of repetitions n ) 3, confidence interval 95%) and filtered again through a 0.45 µm HTTP membrane (Amicon/ Millipore, USA). The final sample had a pH value of 3.9 and an electrical conductivity κ ) 127.8 µS/cm (25 °C). Fractionation and Investigation of Stability over Time. Fractionation of HO12K was performed with an A ¨ kta Explorer 100 HPLC system (Amersham Pharmacia, Sweden) with online UV-absorbance and electrical conductivity detection. The detection wavelength was λ ) 254 nm. A Superdex 75 HR10/30 column by Amersham Pharmacia, Sweden (dextranagarose gel, bed height ca. 300 mm, internal diameter 10 mm) with a bed volume of approximately 24 mL and separation ranges of 3000-70 000 Da (proteins) and 50030 000 Da (dextrans) was used. The composition and the fractionation range of the gel are similar to the widely used Sephadex G-75 sorbent. The exclusion volume Vo (8.5 mL) was determined using blue dextran with a nominal MW of 2 000 000 Da. The permeation volume Vp (18.3 mL) was defined as the negative peak in the electrical conductivity signal of the original sample HO12K which corresponds to VOL. 34, NO. 23, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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the elution volume of the solvent (water). The eluent was a 0.025 mol/L phosphate buffer with an ionic strength of ca. 0.04 mol/L, a pH value of 6.8, and an electrical conductivity of 2.8 mS/cm. The buffer had a DOC concentration of β(DOC) ) 0.3 ( 0.08 mg/L (n ) 3, confidence interval 95%). The flow rate was 1 mL/min. The injection volume was 0.5 mL (2.1% of column volume), and the fraction volume was 1 mL. Twenty-two fractions were collected with an automated fraction collector, starting with fraction 1 (F1) from Ve ) 1.5-2.5 mL. Thirty-six injections were performed subsequently, giving an overall fraction volume of 36 mL. Integration of the UV-absorbance peak areas (PA) was performed using the system-specific control and data evaluation software UNICORN (version 3.00, Amersham Pharmacia Biotech, 1998). The stability of selected fractions over time was also investigated using the A¨ kta system. During the studied time period the fractions were stored in closed containers at 10 °C in the dark. Selected fractions were reinjected after 1 day, and 1, 2, and 5 weeks, respectively. Between the reinjections the column was cleaned with 0.5 N NaOH (1 column volume at a flow rate of 0.5 mL/min). Kd-values were calculated from the elution volumes (Ve) according to Perminova et al. (8) as follows
Kd ) (Ve - Vo)/(Vp - Vo)
(1)
where Kd ) distribution coefficient, Ve ) elution volume in mL, Vo ) exclusion volume in mL, and Vp ) permeation volume in mL. UV peak areas were normalized to the corresponding fraction and injection volumes (V) according to
nPA ) PA/(fraction V/injection V)
(2)
where nPA ) normalized peak area in mAU/mL, PA ) peak area in mAU/mL, and V ) volume in L. Recoveries (R) in % for each fraction F were calculated from the normalized PA:
UV R (F) ) nPA(F)/nPA(HO12K)/100
(3)
The total R in % was defined as the sum of the R of the reinjected fractions. Calibration. To check the performance of the Superdex column and to get an idea of the range of molecular weight of the samples, a set of polystyrene sulfonate standards with peak MWs (Mp) of Mp ) 1370 Da, Mp ) 3800 Da, Mp ) 6710 Da, Mp ) 8000 Da, Mp ) 13 400 Da, and Mp ) 16 900 Da was used for calibration. The standards were prepared in eluent, and the UV-absorbance at a wavelength of λ ) 224 nm was detected. LCDOC Analysis. The LCDOC system with online UVabsorbance (λ ) 254 nm) and DOC-detection according to Huber and Frimmel (12) was employed as an analytical tool to reexamine HO12K and selected fractions. A TSK HW-50 (S) column (Toyopearl, Japan) with a length of 200 mm, an internal diameter of 25 mm, a column volume of 98 mL, and a nominal fractionation range of 100 to 20 000 Da (poly(ethylene glycol)s) was used. TSK-gels of the HW and PW type are hydrophilic copolymers made from ethylene glycol and methacrylate. Vo (22.2 mL) was determined using blue dextran. Vp (46.8 mL) was determined by injection of MilliQ-water, and detection at λ ) 210 nm. Eluent and flowrate were the same as described above. The injection volume was 2 mL (2.0% of column volume). Prior to injection the samples were diluted with eluent to adjust the UV- and DOC-signals to the working range of the detectors. Dilution ratios were sample:eluent ) 1:50 volume/volume (v/v) for HO12K, sample:eluent ) 3:10 v/v for fractions F5-F10, and F16-F18, and sample:eluent ) 1:4 v/v for F11-F15. Peak areas of the UV-absorbance4868
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FIGURE 1. SEC separation of HO12K into 1-mL fractions (Superdex 75 column). Arrows indicate beginning of a new fraction; e.g. F1: Ve ) 1.5-2.5 mL. (el. cond.: electrical conductivity, Ve: elution volume, Vo: exclusion volume, Vp: permeation volume; integration limits shown as tick marks).
TABLE 1. Average Ve- and Kd-Values, and Relative Peak Areas for the Fractionation of HO12K with the A2 kta SEC-Systema HO12K
average Ve in mL
average Kd
relative peak area in %
peak 1 peak 2 peak 3
8.9 ( 0.02 14.8 ( 0.09 17.8 ( 0.02
0.04 ( 0.002 0.64 ( 0.009 0.95 ( 0.002
5 ( 0.7 89 ( 0.9 6 ( 0.2
a
n ) 8, confidence intervals 95%
and DOC-signal were calculated using the software GelTreat (version 1.0, Kudryavtsev, A. V., Moscow State University, 1999). Peak areas of the UV- and DOC-chromatograms were converted to spectral UV-absorbances (UVA) and DOCconcentrations. Normalized UVA- and DOC-values were calculated in analogy to eq 2. UVA and DOC recoveries were then calculated relative to the normalized UVA and DOC signals of HO12K (1:50 v/v) in analogy to eq 3.
Results and Discussion Fractionation and Investigation of Stability over Time. The separation of HO12K into 1-mL fractions can be seen in Figure 1. The sample eluted completely within the separation range of the column (between Vo and Vp), which, according to Swift and Posner (4), indicates that the fractionation was mainly based on molecular size differences. The chromatographic pattern of the sample was highly reproducible which is a prerequisite for an efficient fractionation. Table 1contains the average Ve- and the Kd-values of the detected peaks (see peak labels and integration tick marks in Figure 1) as well as the relative peak areas (% of total peak area) calculated from 8 subsequent fractionation runs of HO12K (n ) 8). The total peak area was 4940 ( 6 mAU/min (95% confidence interval), with a coefficient of variation (CV) of 0.2%. The relative peak areas of the individual peaks varied by less than 1% (95% confidence interval). The CV of the Ve-values was below 1%. Fractions F5-F18 were selected for further studies (reinjection and LCDOC analysis). Reinjection of those fractions on the subsequent day yielded individual peaks in all cases (except F5 and F6 where no UV-signal was detectable). This result is comparable to the one reported by Becher et al. (13) who obtained individual peaks after reinjection of 7 fractions prepared by SEC from original and chlorinated NOM samples. Figure 2a shows the chromatograms of selected fractions reinjected after 1 week (only fractions F8, F10, F12, F13, F15, and F17 shown) and the original signal of HO12K. The chromatographic signals of the fractions not shown here followed the overall trend. The
TABLE 2. Average Ve- and Kd-Values, Asymmetry Factors, and UV Recoveries (R) of the Reinjected Fractions F5-F18 on the A2 kta SEC-System sample
average Ve in mL
F5 n.d. F6 n.d. F7 8.8 ( 0.05 F8 8.9 ( 0.04 F9 11 ( 0.1 F10 12 ( 0.1 F11 13 ( 0.1 F12 13 ( 0.1 F13 14 ( 0.1 F14 15 ( 0.1 F15 16 ( 0.3 F16 17 ( 0.6 F17 18.0 ( 0.03 F18 19 ( 0.1 total R in %
average Kd
asymmetry factor
n.d. n.d. 0.03 ( 0.005 0.04 ( 0.004 0.3 ( 0.01 0.3 ( 0.01 0.4 ( 0.01 0.5 ( 0.01 0.6 ( 0.01 0.7 ( 0.01 0.8 ( 0.03 0.9 ( 0.06 1.00 ( 0.003 1.1 ( 0.01
n.d. n.d. 3(1 11 ( 2 2 ( 0.4 3 ( 0.1 2 ( 0.3 1 ( 0.2 1.0 ( 0.06 0.9 ( 0.03 0.9 ( 0.08 0.8 ( 0.03 0.3 ( 0.06 n.c.
UV R in % 0 0 1 ( 0.3 5 ( 0.5 6 ( 0.7 9 ( 0.7 12 ( 0.2 14 ( 0.4 15 ( 0.4 14 ( 0.5 10 ( 5 5 ( 0.7 2 ( 0.5 0.1 ( 0.02 90 ( 4
a n ) 4, confidence intervals 95%, n.d.: not detectable, n.c.: not calculated.
FIGURE 2. Chromatograms of the original sample HO12K and selected fractions (F8, F10, F12, F13, F15, and F17, Superdex 75 column, reinjection after 1 week): (a) UV-signals of selected fractions and HO12K and (b) sum of UV-signal intensities of all fractions, original UV-signal of HO12K, and corresponding difference signal. individual peaks fit well under the HO12K curve and collectively appear similar to the original chromatogram. However, it is also obvious that some material must have been lost since the chromatograms of the fractions do not follow the original curve exactly. This can also be seen in Figure 2b, where the sum of the UV-signal intensities of all fractions is compared to the original chromatogram of HO12K. The difference signal of the two curves reveals that mainly material in the elution range Ve ) 8-14 mL was lost. The discrepancy can be ascribed to hydrophobic material which adsorbed irreversibly onto the gel phase. The Ve and the peak areas of the reinjected fractions did not change significantly throughout the study time period (Table 2). Average values and 95% confidence intervals (CI) of the Ve- and Kd-values as well as of the peak asymmetry factors (AF) and UV recoveries of all 4 reinjections (n ) 4) are reported. A very good linear correlation between the average Kd-values (y) and the nominal Kd-values (x) of the fractions F7-F18 was obtained (y ) 0.9386x + 0.0671; R 2 ) 0.9937; figure not shown). The nominal Ve was defined as the average of the Ve at which the collection of a particular fraction was started (Ve(start)) and the Ve at which the collection was ended (Ve(end)):
nominal Ve ) (Ve(start) + Ve(end))/2
(4)
A slope close to one indicates that all reinjected fractions eluted near the original Ve and indicates that the majority of the compounds in a particular fraction were of a similar molecular size. The CI of the Ve and hence also of the Kdvalues of some of the fractions are greater than the exceptionally low CI reported for HO12K, but are still within
an acceptable range. The CI of the UV recoveries were for all fractions except F15 within the reproducibility limits which means that no significant changes in the sample composition and concentration occurred. The deviations in the case of F15 are due to an incomplete sample injection during the second reinjection. The total recovery was consistently around 90% with a maximum of 15 ( 0.4% in F13. The fact that all fractions were very stable also suggests that solutes of a similar size could be collected in each fraction and that those fractions did not undergo major changes in their size or conformation over a time period of 5 weeks. In other words, no reorganization regarding molecular size could be observed. If the separation was the result of ideal SEC behavior, however, the individual peaks should all have a more or less perfect Gaussian shape and elute near the Ve where they were collected originally. The peak shape can be described using the asymmetry factor (AF) which was calculated according to
AF ) a/b
(5)
where a and b are the partial peak widths measured at 10% peak height. As can be seen from the asymmetry factors reported in Table 2, only the fractions F12 and F13 have the typical Gaussian peaks that would be expected (AF ≈ 1). In contrast, the peaks of F7-F11 have a distinct tailing which gradually transforms into Gaussian peaks and finally into peaks with a clear fronting in the case of F16 and F17. The reasons for this behavior are not completely clear, but nonspecific interactions definitely play a role. The observed phenomena can be a consequence of adsorption mechanisms or ionic interactions which could have been present during fractionation, reinjection, or during both. Since those mechanisms depend on the sample concentration and on the pH and the ionic strength of the sample and the eluent, it is important to consider those parameters. A possible explanation for the peak shapes is that they are a consequence of nonspecific interactions during the original fractionation procedure. It is reasonable to assume that during the fractionation, a particular parameter (e.g. pH) was changing gradually which could have resulted in a gradual transformation from adsorptive to ionic interactions, or vice versa. Thus, a selective retention of solutes regarding their tendency to participate in either one of the mechanisms would take place in addition to the separation according to molecular size. The separation would then lead to fractions VOL. 34, NO. 23, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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of different molecular sizes and/or chemical-physical properties. During the subsequent reinjection this gradual change would be absent, and the solutes would elute according to their MW. To examine this hypothesis HO12K was analyzed again under the same SEC conditions, but with additional online pH-monitoring. It was found that in spite of the injection of an acidic sample the pH remained constant throughout the run except for an increase by 0.3 pH units at Ve ) 18.3 mL (Vp) and a decrease by 0.4 pH units at Ve ) 19.5 mL. Therefore, pH cannot be the determining parameter for a gradual change in the separation mechanisms. The same holds true for the ionic strength, which can be approximated through the electrical conductivity. No gradual change in the electrical conductivity was detectable throughout the fractionation (except for the negative peak at Vp). Nonspecific interactions could also have been present during the reinjection. To evaluate this case, the pH-value and the ionic strength of the fractions must be considered. F5-F18 all had the same pH-value of 6.8, and an electrical conductivity κ ) 2.8 mS/cm (values identical to those of the eluent). Those parameters therefore cannot have resulted in fractions of different peak shapes. The particular peak forms must thus be a consequence of differences in the composition or the concentration of the individual fractions. The composition of each fraction cannot be determined exactly. During fractionation the very high sample concentration could have resulted in an overloading of the column, and a breakthrough of small-sized molecules so that the fractions collected at an early Ve could also contain proportions of molecules with lower MW. During reinjection of the less concentrated fractions no overloading should have occurred so that the small-sized molecules eluted according to their MW and resulted in peak tailing. Another possibility is that during fractionation as well as during reinjection a proportion of solutes, especially of solutes with a high MW which are more susceptible to van der Waals forces, adsorbed reversibly onto the gel phase and were retarded. If it is assumed that the number of sorption sites on the gel remains constant, the relative amount of adsorbed solutes would increase with decreasing sample concentration. Therefore this effect should be much more noticeable in the chromatograms of the fractions than in the chromatogram of HO12K and result in peak tailing. An analogous explanation can be given for the fronting observed for F16 and F17. Those fractions should mainly consist of small molecules (according to SEC theory) which, however, can still participate in nonideal mechanisms. In this case the charge density of the solutes is of interest. According to Perminova et al. (8) the charge density is defined as the number of ionogenic groups per mole divided by the molecular weight * 1000. Hence it is especially important for small molecules which have a low MW. The effect is also closely related to the number of ionic sites of the gel phase. Since this type of interaction is only efficient over short distances, and since the number of ionogenic sites of the gel phase is limited at high sample concentrations only a small proportion of the solutes will be affected. At low sample concentrations this proportion will be greater, and molecules with a high charge density will be overexcluded and cause peak fronting. To determine which of the discussed possibilities are warranted further studies, especially on the functionality of the fractions, are needed. Indications for susceptibility of the original sample itself for nonspecific interactions can be obtained from data on its chemical-physical properties such as the elemental composition or proton capacity. Conclusions drawn from those data can only be general trends for the overall sample, though, and they may not apply for the individual molecules present. For the original sample HO12K, as reported by Frimmel and Abbt-Braun (11), the elemental analysis of the sample yielded atomic ratios of approximately 4870
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FIGURE 3. Chromatograms of HO12K (1:50 v/v) on the two SECsystems as a function of Kd (A2 kta-SEC system: Superdex 75 column, and LCDOC-SEC system: TSK HW-50 (S) column). H/C ) 0.9, O/C ) 0.6, and N/C ) 0.02. The H/C ratio is rather low and indicates a high content of aromatic structures, and hence a relatively hydrophobic character. This interpretation is supported by the fairly high specific UV-absorbance (SUVA) of HO12K. The SUVA is defined as the ratio of the spectral UV-absorbance (UVA) to the DOC-concentration. It provides information on the density of double bonds which can be found in certain functional groups and in aromatic systems. The membrane-concentrated sample HO12K had a SUVA of approximately 5.2 L/mg/m, whereas an original sample from the same site had a SUVA of ca. 4.4 L/mg/m (11). This indicates that, compared to the original sample, HO12K is enriched in UV-active, or aromatic (hydrophobic) compounds. As reported by Perminova et al. (8) and others the hydrophobic character of the solutes can result in their adsorption onto hydrophobic structures in the gel phase. The specific proton capacities of HO12K were reported to be 5.2 µmol/mg (DOC) for pH < 7 (carboxylic structures) and 2.9 µmol/mg (DOC) for pH > 7 (phenolic structures) (11). The former values are fairly low when compared to fulvic acids from the same sampling site which means that the sample contains only a small number of ionizable groups. It could therefore be concluded that for the original sample HO12K electrostatic interactions should be of minor importance. Calibration. The relationship between the logarithms of the Mp and the Kd of the PSS standards was log Mp ) -1.3477/Kd + 4.3006. The correlation coefficient was R 2 ) 0.9471. The Mp derived from eq 4 ranged from Mp ) 17 870 ( 113 Da to Mp ) 799 ( 28 Da for the original sample HO12K and from Mp ) 17 998 ( 306 Da to Mp ) 737 ( 30 Da for the selected fractions which is within the range of MW values reported for NOM in the literature (8, 13, 14). LCDOC Analysis and Comparison of the Two SEC Systems. In Figure 3 the chromatograms of HO12K (1:50 v/v) analyzed with the A¨ kta and the LCDOC system are shown as a function of the Kd-values. The overall shape of the chromatogram was similar with both systems. From the Kd-values it can be seen, however, that with the TSK HW-50 (S) column the sample eluted much closer to the exclusion limit than with the Superdex column. The lower upper separation range reported for the TSK HW-50 (S) column explains this observation. The distribution of the solutes also varied with the column type as can be derived from the relative peak areas. Hence, the peak shape is also a function of the column material. Fractions F5-F18 were also analyzed with the LCDOC system. A similar distribution with individual Gaussian-type peaks was obtained for both the UV- and the DOC-signal. The DOC-signals of selected fractions (same fractions as in Figure 2a)) are shown in Figure 4. The fact that individual
FIGURE 5. Spectral UV-absorbances (UVA), DOC-concentrations, and SUVA-values of fractions F5-F18 (TSK HW-50 (S) column). FIGURE 4. DOC-Signals of selected fractions and HO12K (1:50 v/v) (TSK HW-50 (S) column, reinjection after 1 week.
TABLE 3. Kd-Values and UVA and DOC Recoveries (R) of the Reinjected Fractions F5-F18 on the LCDOC-SEC-System sample
Kd
UVA R in %
DOC R in %
F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17 F18 total R in %
n.d n.d. 0 0 0.2 0.2 0.2 0.3 0.4 0.5 0.5 0.6 1.1 n.d.
0 0 1 6 8 11 14 14 15 15 9 5 3 0 102
0 0 4 7 7 8 11 15 16 17 13 6 3 0 105
a
n ) 1. n.d.: not detectable.
peaks of a Gaussian type shape could even be obtained with a different column material supports the hypothesis that fractions of a narrow molecular size range could be produced and that molecular size was the dominating factor for the separation. If the chromatographic behavior was mainly a result of nonspecific interactions, two different stationary phases should result in clearly different chromatograms for the same sample. As was observed for the HO12K (1:50 v/v) sample, the LCDOC Kd-values of the fractions were also lower than the A¨ kta-Kd-values due to the lower separation range of the TSK column. Kd-values and UVA and DOC R calculated from column measurements are reported in Table 3. The total recoveries were 102% for the UVA and 105% for the DOC mass balance. It is interesting that both recoveries were very similar and above 100%. For the DOC this can easily be explained if the DOC content of the eluent is considered. The DOC mass contained in the total volume of F5-F18 corresponds to a DOC recovery of about 5% which means that the net total DOC R is about 100%. When the UV and UVA recoveries obtained with the two systems are compared it must be considered that the sample HO12K had to be diluted 50-fold to a β(DOC) ) 3.6 mg/L for the LCDOC analysis. This affected the so-called “hUVA” (hydrophobic UV-absorbing solutes) which can be defined as the proportion of the injected sample that is lost due to irreversible adsorption onto the column material. The relative hUVA in % can be calculated from bypass versus column measurements according to
hUVA ) 100 - (PA column/PA bypass/100)
(6)
For the Superdex column it was found that the hUVA
increased as a function of decreasing sample concentration of HO12K from 16% for HO12K (1:2 v/v) to 34% for HO12K (1:83 v/v). This tendency should also hold true for the TSK HW-50 (S) column. On the latter column losses due to adsorption generally range from 10% to 20% of the DOC mass injected for this type of sample. Thus the higher hUVA in the case of the HO12K (1:50 v/v) sample results in a different, relatively lower 100%-basis for the calculation of the UV R. Since several of the fractions had a higher DOC, and hence a lower hUVA than HO12K (1:50 v/v), the total R therefore can amount to more than 100%. Despite this, the UVA R determined with the LCDOC system were similar to the A¨ kta UV R and confirm the results obtained with the latter SEC system. The maximum UVA R, which corresponds to a UVA of 91 1/m, was detected in F13. The spectral UV-absorbance data (UVA), the DOCconcentrations, and the SUVA-values of the fractions F5F18 analyzed with the LCDOC system are shown in Figure 5. The maximum of the DOC concentration was found in F14 (β(DOC) ) 15.3 mg/L, or 17%). The SUVA-values were highest in F10 (SUVA ) 8.9 L/mg/m). Thus, in this fraction the UV-absorbing structures are most abundant. A high SUVA was also found in F17 (SUVA ) 6.7 L/mg/m). The average DOC-normalized UVA of all fractions was SUVA ) 4.8 L/mg/m (pH ) 6.8) and substantially lower than the SUVA of HO12K (6.4 L/mg/m at pH ) 3.9) despite the considerably higher pH of the fractions. This also indicates that solutes with strong UV-absorbance were lost due to irreversible adsorption and that the UV-absorbing acitivity of those compounds was to a great extent caused by aromatic subunits, which are prone to adsorb well. Overall, within the analytical limitations, both SEC systems yielded comparable UV recoveries. The LCDOC analysis therefore confirmed the A¨ kta results and supplied the additional information that the total DOC recovery was in the same range. Both columns differed in their separation efficiency with the Superdex column performing better than the TSK HW-50 S column in the high MW range. The separation ranges for compounds with linear structures (dextrans, PEG) given by the manufacturers help to explain this observation.
Acknowledgments This work was financially supported by the German Research Foundation (DFG). The authors would like to thank Gabi Kolliopoulos for the thorough performance of the LCDOC measurements and Gudrun Abbt-Braun and Christian Zwiener for valuable discussions and additional information.
Literature Cited (1) Swift, R. S. In Humic Substances in Soil, Sediment, and Water; Aiken, G. R., McKnight, D. M., Wershaw, R. L., Eds.; Wiley: New York, 1989; pp 387-408. (2) Gelotte, B. J. Chrom. 1959, 3, 330. (3) Lindqvist, I. Acta Chem. Scand. 1967, 21, 2564. VOL. 34, NO. 23, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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(4) (5) (6) (7) (8) (9) (10) (11) (12) (13)
Swift, R. W.; Posner, A. M. J. Soil Sci. 1971, 22, 237. Aho, J.; Lehto, O. Arch. Hydrobiol. 1984, 101, 21. Fuchs F. Vom Wasser 1985, 64, 129. Specht, C. H.; Frimmel, F. H. Environ. Sci. Technol. 2000, 34, 2361. Perminova, I. V.; Frimmel, F. H.; Kovalevskii, D. V.; Abbt-Braun, G.; Kudryavtsev; A. V.; Hesse, S Water Res. 1998, 32, 872. Peuravuori, J., Pihlaja, K. Anal. Chim. Acta 1997, 337, 133. Pelekani, C.; Newcombe, G.; Snoeyink, V. L.; Hepplewhite, C.; Assemi, S.; Beckett, R. Environ. Sci. Technol. 1999, 33, 2807. Frimmel, F. H.; Abbt-Braun, G. Environ. Internat. 1999, 25, 191. Huber, S.; Frimmel, F. H. Anal. Chem. 1991, 63, 2122. Becher, G.; Carlberg, G. E.; Gjessing, E. T.; Hongslo, J. K.; Monarca, S. Environ. Sci. Technol. 1985, 19, 422.
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(14) Amy, G. L.; Collins, M. R.; Kuo, C. J.; King, P. H. J. Am. Wat. Works Assoc. 1987, 79, 43. (15) Wershaw, R. L.; Aiken, G. R. In Humic Substances in Soil, Sediment, and Water; Aiken, G. R., McKnight, D. M., Wershaw, R. L., Eds.; Wiley: New York, 1989; pp 477-492.
Received for review April 3, 2000. Revised manuscript received August 22, 2000. Accepted August 30, 2000.
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